Cells synthesize glucose if deprived of it, and destroy gluconeogenic enzymes upon return to
glucose-replete conditions. We found that the Gid4 subunit of the ubiquitin ligase GID in the
yeast Saccharomyces cerevisiae targeted the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 for
degradation. Gid4 recognized the N-terminal proline (Pro) residue and the ~5-residue-long
adjacent sequence motifs. Pck1, the fourth gluconeogenic enzyme, contains Pro at position 2;

Gid4 directly or indirectly recognized Pro at position 2 of Pck1, contributing to its targeting.

These and related results identified Gid4 as the recognition component of the GID-based
proteolytic system termed the Pro/N-end rule pathway. Substrates of this pathway include
gluconeogenic enzymes that bear either the N-terminal Pro residue or a Pro at position 2,
together with adjacent sequence motifs.

When glucose is low or absent, cells syn- thesize it through gluconeogenesis, an adenosine triphosphate (ATP)–consuming process. Gluconeogenesis is, in effect, a reversal of glycolysis, in which glucose
is converted to pyruvate, with production of ATP
and reduced nicotinamide adenine dinucleotide
(NADH) (fig. S1). Some enzymatic steps are shared
between gluconeogenesis and glycolysis, but other
steps are confined to one of the two pathways (1, 2).
In the yeast S. cerevisiae, the main gluconeogenesis-specific enzymes are fructose-1,6-bisphosphatase
(Fbp1), isocitrate lyase (Icl1), malate dehydrogenase
(Mdh2), and phosphoenolpyruvate carboxykinase
(Pck1) (fig. S1) (3–12). Mammalian counterparts
of these enzymes are regulated by insulin and
other hormones, and become essential for viability
during fasting. Aberrant control of gluconeogenic enzymes can promote cancer, diabetes,
and other human diseases (13, 14).

When S. cerevisiae grows on ethanol or acetate, the gluconeogenic enzymes are expressed
and long-lived. Transition to a medium containing
glucose inhibits the synthesis of these enzymes
and induces their degradation (fig. S2, A and B).
Screens for mutants unable to destroy Fbp1 identified subunits of the ubiquitin (Ub) ligase termed
GID (glucose-induced degradation) (3, 6–8). The
GID Ub ligase of S. cerevisiae contains eight distinct subunits and is conserved among eukaryotes,
including animals and plants (5, 6, 15–17).

A 1998 study by Wolf and colleagues suggestedthat the GID/proteasome-dependent degradationof Fbp1, Icl1, and Mdh2—three of the four yeastgluconeogenic enzymes—may be mediated bytheir common property of bearing the N-terminalproline (Pro) residue (4). We decided to inves-tigate this suggestion and found that Gid4 (alsoknown as Vid24) is a substrate-targeting sub-unit of the GID Ub ligase.

The N-end rule pathway is a set of proteolytic
systems whose unifying feature is their ability to
recognize and polyubiquitylate proteins containing
N-terminal degradation signals called N-degrons,
thereby causing the degradation of these proteins
by the proteasome (18–27). The main determinant
of an N-degron is a destabilizing N-terminal residue
of a protein. Recognition components of the N-end
rule pathway, called N-recognins, are E3 Ub ligases
that can target N-degrons (Fig. 1).

Regulated degradation of proteins and their
natural fragments by the N-end rule pathway mediates a remarkably broad range of biological processes, including the sensing of heme, O2, NO, and
short peptides; the control of subunit stoichiometries in oligomeric proteins; the elimination
of misfolded and otherwise abnormal proteins; the
degradation of proteins after their translocation
to the cytosol from membrane-enclosed compartments; regulation of apoptosis and repression of
neurodegeneration; regulation of DNA repair, transcription, replication, and chromosome cohesion/
segregation; regulation of G proteins, cytoskeletal
proteins, autophagy, peptide import, meiosis, immunity, circadian rhythms, fat metabolism, cell
migration, cardiovascular development, spermatogenesis, and neurogenesis; the functioning of adult
organs, including the brain, muscle, testis, and
pancreas; and the regulation of leaf and shoot
development, senescence, NO/O2 sensing, and
many other processes in plants (22–29).

The pathway’s other branch, called the Ac/N-end rule pathway, targets proteins for degradation
by recognizing their Nt-acetylated residues (Fig.
1D) (19, 20, 30, 38–40). N-degrons and Ub ligases of
the Ac/N-end rule pathway are called Ac/N-degrons
and Ac/N-recognins, respectively. At least 60% of S.
cerevisiae proteins and about 90% of human proteins are cotranslationally and irreversibly Nt-acetylated by ribosome-associated Nt-acetylases

We began by designing a version of the promoter
reference technique (PRT) (Fig. 2, A and B). In
this method, one measures, during a chase, the
ratio of a test protein to a long-lived reference protein, the mouse dihydrofolate reductase (DHFR).
Epitope-tagged reference and test proteins were
coexpressed from two identical PTDH3 promoters
containing additional DNA elements developed
by others (41). Once transcribed into an mRNA,
these elements form 5′-RNA aptamers that can
bind to tetracycline, thereby repressing translation in cis (Fig. 2B). This PRT design improves
degradation assays by providing a reference and
by allowing chases that involve a gene-specific
repression of translation. The latter option avoids
a global shutoff of translation by inhibitors such
as cycloheximide. This advantage is particularly
important if an essential component of a relevant
proteolytic pathway is itself short-lived.

After 18 hours in ethanol medium at 30°C,
S. cerevisiae were shifted to a glucose medium
while the synthesis of the 348-residue, C-terminally
Flag-tagged P-Fbp13f (bearing N-terminal Pro) and
the coexpressed 187-residue DHFR reference was
repressed by tetracycline, initiating a chase (Fig. 2,
C and D). Under these conditions, P-Fbp13f [its N-terminal Met is cotranslationally removed by Met-aminopeptidases (42)] was short-lived in wild-type
cells (t1/2 < 30 min) but was stable in gid1D, gid2D,
gid3D, and gid4D mutants that lacked specific subunits of the GID Ub ligase (Fig. 1C, Fig. 2, C and D,
and fig. S3, A and C). In contrast, the S-Fbp13f
mutant, bearing N-terminal Ser instead of Pro, was
long-lived even in wild-type cells (Fig. 2, C and D).
Degradation assays with another gluconeogenic
enzyme, the 377-residue cytosolic P-Mdh23f and
its S-Mdh23f mutant (bearing N-terminal Ser),
produced similar results (Fig. 2, E and F).

Gid4 as a recognition subunit of GID

Given these findings, we wished to identify an
N-recognin of the GID Ub ligase. The successful